The goal of this proposal is to study how hepatocytes control the number, shape, and distribution of mitochondria. It is well known that mitochondria perform vital cellular functions in the hepatocyte including ATP generation, calcium homeostasis, regulation of redox state, and participation in signaling cascades that can regulate cell death. While mitochondrial dynamics appear to play an important role in these processes, the molecular mechanisms by which these essential organelles change their form and cytoplasmic location are poorly understood. Recently, we and others have identified two large GTPases, DLP1 and Mfn, which are found to participate in maintaining normal mitochondrial morphology in yeast and rat hepatocytes by mediating membrane fission and fusion. DLP1 is a dynamin-like protein that is presumed to function in mitochondrial division via its GTPase activity while Mfn has been reported to be a major factor involved in mitochondrial fusion. Our published work and substantial preliminary data in hepatocytes support the central hypothesis of this proposal that hepatic mitochondrial morphology and function is maintained through the antagonistic action of two large GTPases, DLP1 and Mfn. This proposal describes novel in vivo and in vitro studies, combined with numerous molecular and immunological reagents that we have made, to define the role of DLP1 and Mfn in regulating mitochondrial shape in the hepatocyte and to provide important insights into how these large GTPases function. We will pursue three Specific Aims. First, using state-of-the-art microscopic imaging techniques, we will test the respective roles of DLP1 and Mfn in maintaining mitochondrial number and morphology in living hepatocytes. Second, structural studies will define, at a molecular level, how DLP1 acts to sever mitochondrial membranes and whether this severing activity is enhanced by putative binding partners that we have already identified. Third, we will define how cellular signaling cascades regulate DLP1 and Mfn activity to subsequently alter the form and function of hepatocyte mitochondria. Information gathered from these studies will greatly increase our understanding of how mitochondrial shape and number are regulated and contribute to hepatocellular physiology and injury in health and disease.